CN115006600A - 具有生物活性的温敏角膜修复水凝胶制备方法及其应用 - Google Patents
具有生物活性的温敏角膜修复水凝胶制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种具有生物活性的温敏角膜修复水凝胶的制备方法,步骤如下:S1、收集生物组织;S2、病毒灭活;S3、反复冻融;S4、组织脱细胞;S5、制备凝胶:将步骤S4获得的组织先置于‑100℃~‑40℃预冻12~18h,冻干;再将冻干组织研磨成小于1mm的脱细胞颗粒,进行酶解,完成后用碱液调节pH至6.0~7.5,中和等渗溶液,并调节浓度;S6、添加防腐体系,即可。本发明采用上述的一种具有生物活性的温敏角膜修复水凝胶制备方法及其应用,制备的水凝胶中含有丰富胶原蛋白、弹性蛋白和粘多糖,有效抑制物理或化学损伤造成的角膜病变进程,并在体温条件下自交联形成“生物膜”起到物体屏障的作用,从而促进受损角膜再生修复。
Description
技术领域
本发明涉及生物组织工程技术领域,尤其是涉及一种具有生物活性的温敏角膜修复水凝胶制备方法及其应用。
背景技术
角膜是一种透明组织,位于眼球前壁,主要行使屏障和屈光功能。由于角膜直接与外界接触,极易受到机械外伤、热灼伤、酸碱侵蚀等,从而引起角膜疾病的发生,严重者甚至失明。目前,大多数角膜疾病通过移植手术脱盲,然而,若缺乏角膜供体而无法通过角膜移植手术脱盲。
随着科技的进步,角膜修复技术逐渐发展。其中,“膜型”角膜修复,由于材料的特殊性,需要通过手术缝合才能实现,使用繁琐,不利于日常使用。另外,在专利中,CN201610557778.1具有生物活性的角膜修复植片的制备方法和角膜修复植片公开了角膜修复水凝胶,其交联过程是在使用前完成,这会造成使用时产品在角膜处无法均匀铺展。
发明内容
本发明的目的是提供一种具有生物活性的温敏角膜修复水凝胶制备方法及其应用,有效抑制物理或化学损伤造成的角膜病变进程,并在体温条件下自交联形成“生物膜”起到物体屏障的作用,从而促进受损角膜再生修复。
为实现上述目的,本发明提供了一种具有生物活性的温敏角膜修复水凝胶的制备方法,步骤如下:
S1、收集生物组织;
S2、病毒灭活:
将收集到的新鲜组织,剪成小块后,添加病毒灭活工作液,并于室温浸泡后,捞出;用纯化水反复清洗,直至洗脱液pH呈中性;
S3、反复冻融;
S4、组织脱细胞:
将步骤S3获得的组织依次浸泡于脱细胞工作液Ⅰ、脱细胞工作液Ⅱ和脱细胞工作液Ⅲ中各2~4h,捞出后用纯化水清洗3~5遍,每次0.5~2h;随后浸泡于脱细胞工作液Ⅳ中4~10h,捞出后用纯化水清洗3~5遍,每次0.5~2h;
S5、制备凝胶:
将步骤S4获得的组织先置于-100℃~-40℃预冻12~18h,冻干;再将冻干组织研磨成小于1mm的脱细胞颗粒,进行酶解,完成后用碱液调节pH至6.0~7.5,中和等渗溶液,并调节浓度,使得最终等渗溶液浓度为2~8mg/mL;
S6、添加防腐体系,即可。
优选的,步骤S1中,组织包括但不限于羊膜、胎盘、皮肤、角膜、小肠下粘膜、膀胱膜;组织来源包括但不限于人、猪、牛、羊、马。
优选的,步骤S1中,组织需要在活体剥离6h内收集完毕。
优选的,步骤S1中,组织收集的过程中温度条件为-20℃~-8℃。
优选的,步骤S2中,病毒灭活工作液由过氧乙酸:无水乙醇:纯化水按照体积比3:5:92配制。
优选的,步骤S2中,病毒灭活的过程为将收集到的新鲜组织,剪成2cm×2cm小块,然后按照1kg组织:10L病毒灭活工作液的比例混合,室温20℃~30℃浸泡0.5~4h后,捞出灭活后的组织,用纯化水反复清洗,直至洗脱液pH为6.0~7.5。
优选的,步骤S3中,反复冻融的过程为在-100℃~-40℃条件下冷冻1~7天,然后于20℃~30℃条件下溶解,反复3~5次,有效降低组织免疫原性。
优选的,步骤S4中,脱细胞的温度为20℃~30℃,脱细胞工作液Ⅰ为按质量百分比为0.1%~1%胰蛋白酶、0.01%~0.1%乙二胺四乙酸四钠(EDTA-4Na)和0.1%~1%氯化钠的溶液;
脱细胞工作液Ⅱ为质量百分比为1%~5%聚乙二醇辛基苯基醚(Triton X-100)的溶液;
脱细胞工作液Ⅲ为质量百分比为0.5%~5%脱氧胆酸钠的溶液;
脱细胞工作液Ⅳ为质量百分比为0.01%~3%过氧乙酸、1%~8%乙醇的溶液。
优选的,步骤S5中酶解时,添加比例为10mg脱细胞颗粒:1mg胃蛋白酶:1mL 0.01M盐酸溶液,使得脱细胞颗粒最终浓度为10mg/mL,消化15~30h。
优选的,步骤S6中,防腐体系为0.1%~0.5%无水三氯叔丁醇、0.1%~0.3%羟苯乙酯、0.015-0.2%羟苯甲酯、0.002%~0.01%苯扎氯氨中的任一种。
因此,本发明采用上述一种具有生物活性的温敏角膜修复水凝胶制备方法及其应用,制备的水凝胶中含有丰富胶原蛋白、弹性蛋白和粘多糖,有效抑制物理或化学损伤造成的角膜病变进程,并在体温条件下自交联形成“生物膜”起到物体屏障的作用,从而促进受损角膜再生修复。
本发明所记载技术方案的具体技术效果如下:
(1)本发明的角膜修复水凝胶具有细胞生长及组织再生所需营养物质,成分丰富,有利于组织快速修复。
(2)本发明的角膜修复水凝胶以液态形式使用,更加便捷,并且添加防腐体系,更加安全。
(3)本发明的角膜修复水凝胶具有温敏性,在室温为液态,接触眼表后发生自交联,可均匀涂布眼表角膜并形成物理屏障,促进组织再生。
下面通过附图和实施例,对本发明的技术方案做进一步的详细描述。
附图说明
图1是本发明一种具有生物活性的温敏角膜修复水凝胶制备方法及其应用的流程图。
具体实施方式
以下通过实施例对本发明的技术方案作进一步说明。
除非另外定义,本发明使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的主旨或基本特征的情况下,能够以其它的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内,不应将权利要求视为限制所涉及的权利要求。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其它实施方式。这些其它实施方式也涵盖在本发明的保护范围内。
还应当理解,以上所述的具体实施例仅用于解释本发明,本发明的保护范围并不限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明/发明的保护范围之内。
本发明中使用的“包括”或者“包含”等类似的词语意指在该词前的要素涵盖在该词后列举的要素,并不排除也涵盖其它要素的可能。术语“内”、“外”、“上”、“下”等指示的方位或位置关系为基于附图所示的方位或位置关系,仅是为了便于描述本发明和简化描述,而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作,因此不能理解为对本发明的限制,当被描述对象的绝对位置改变后,则该相对位置关系也可能相应地改变。在本发明中,除非另有明确的规定和限定,术语“附着”等术语应做广义理解,例如,可以是固定连接,也可以是可拆卸连接,或成一体;可以是直接相连,也可以通过中间媒介间接相连,可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言,可以根据具体情况理解上述术语在本发明中的具体含义。本发明中使用的术语“约”具有本领域技术人员公知的含义,优选指该术语所修饰的数值在其±50%,±40%,±30%,±20%,±10%,±5%或±1%范围内。
本公开使用的所有术语(包括技术术语或者科学术语)与本公开所属领域的普通技术人员理解的含义相同,除非另外特别定义。还应当理解,在诸如通用词典中定义的术语应当被理解为具有与它们在相关技术的上下文中的含义相一致的含义,而不应用理想化或极度形式化的意义来解释,除非本文有明确地这样定义。
对于相关领域普通技术人员已知的技术、方法和设备可能不作为详细讨论,但在适当情况下,所述技术、方法和设备应当被视为说明书的一部分。
本发明说明书中引用的现有技术文献所公开的内容整体均通过引用并入本发明中,并且因此是本发明公开内容的一部分。
实施例一
一种具有生物活性的温敏角膜修复水凝胶的制备方法,步骤如下:
S1、收集生物组织;收集6h内剥离的胎盘,在组织收集的过程中温度条件为-8℃。
S2、病毒灭活:
将收集到的新鲜组织,剪成2cm×2cm小块,然后按照1kg组织:10L病毒灭活工作液的比例混合,室温浸泡1h后,捞出灭活后的组织,用纯化水反复清洗,直至洗脱液pH为6.5。病毒灭活工作液由过氧乙酸:无水乙醇:纯化水按照体积比3:5:92配制。
S3、反复冻融;反复冻融的过程为在-80℃条件下冷冻2天,然后于25℃条件下溶解,反复3次,有效降低组织免疫原性。
S4、组织脱细胞:
脱细胞于室温中进行,将步骤S3获得的组织依次浸泡于脱细胞工作液Ⅰ、脱细胞工作液Ⅱ和脱细胞工作液Ⅲ中各2h,捞出后用纯化水清洗3遍,每次1h;随后浸泡于脱细胞工作液Ⅳ中5h,捞出后用纯化水清洗3遍,每次0.5h;
脱细胞工作液Ⅰ为按质量百分比为0.2%胰蛋白酶、0.03%乙二胺四乙酸四钠(EDTA-4Na)和0.5%氯化钠的溶液;脱细胞工作液Ⅱ为质量百分比为3%聚乙二醇辛基苯基醚(Triton X-100)的溶液;脱细胞工作液Ⅲ为质量百分比为2%脱氧胆酸钠的溶液;脱细胞工作液Ⅳ为质量百分比为0.12%过氧乙酸、3%乙醇的溶液。
S5、制备凝胶:
将步骤S4获得的组织先置于-80℃预冻12h,冻干;再将冻干组织研磨成小于1mm的脱细胞颗粒,进行酶解,酶解时配比为10mg脱细胞颗粒:1mg胃蛋白酶:1mL 0.01M盐酸溶液,使得脱细胞颗粒最终浓度为10mg/mL,消化15~30h。完成后用碱液调节pH至6.5,中和等渗溶液,并调节浓度,使得最终等渗溶液浓度为4mg/mL。
S6、添加防腐体系,防腐体系为0.2%无水三氯叔丁醇。
将实施例一制得的凝胶直接涂布于眼中,即可。
因此,本发明采用上述一种具有生物活性的温敏角膜修复水凝胶制备方法及其应用,有效抑制物理或化学损伤造成的角膜病变进程,并在体温条件下自交联形成“生物膜”起到物体屏障的作用,从而促进受损角膜再生修复。
最后应说明的是:以上实施例仅用以说明本发明的技术方案而非对其进行限制,尽管参照较佳实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对本发明的技术方案进行修改或者等同替换,而这些修改或者等同替换亦不能使修改后的技术方案脱离本发明技术方案的精神和范围。
Claims (10)
1.一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于,步骤如下:
S1、收集生物组织;
S2、病毒灭活:
将收集到的新鲜组织,剪成小块后,添加病毒灭活工作液,并于室温浸泡后,捞出;用纯化水反复清洗,直至洗脱液pH呈中性;
S3、反复冻融;
S4、组织脱细胞:
将步骤S3获得的组织依次浸泡于脱细胞工作液Ⅰ、脱细胞工作液Ⅱ和脱细胞工作液Ⅲ中各2~4h,捞出后用纯化水清洗3~5遍,每次0.5~2h;随后浸泡于脱细胞工作液Ⅳ中4~10h,捞出后用纯化水清洗3~5遍,每次0.5~2h;
S5、制备凝胶:
将步骤S4获得的组织先置于-100℃~-40℃预冻12~18h,冻干;再将冻干组织研磨成小于1mm的脱细胞颗粒,进行酶解,完成后用碱液调节pH至6.0~7.5,中和等渗溶液,并调节浓度,使得最终等渗溶液浓度为2~8mg/mL;
S6、添加防腐体系,即可。
2.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S1中,组织包括但不限于羊膜、胎盘、皮肤、角膜、小肠下粘膜、膀胱膜;组织来源包括但不限于人、猪、牛、羊、马。
3.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S1中,组织需要在活体剥离6h内收集完毕。
4.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S1中,组织收集的过程中温度条件为-20℃~-8℃。
5.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S2中,病毒灭活工作液由过氧乙酸:无水乙醇:纯化水按照体积比3:5:92配制。
6.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S2中,病毒灭活的过程为将收集到的新鲜组织,剪成2cm×2cm小块,然后按照1kg组织:10L病毒灭活工作液的比例混合,室温20℃~30℃浸泡0.5~4h后,捞出灭活后的组织,用纯化水反复清洗,直至洗脱液pH为6.0~7.5。
7.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S3中,反复冻融的过程为在-100℃~-40℃条件下冷冻1~7天,然后于20℃~30℃条件下溶解,反复3~5次,有效降低组织免疫原性。
8.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S4中,脱细胞的温度为20℃~30℃,脱细胞工作液Ⅰ为按质量百分比为0.1%~1%胰蛋白酶、0.01%~0.1%乙二胺四乙酸四钠(EDTA-4Na)和0.1%~1%氯化钠的溶液;
脱细胞工作液Ⅱ为质量百分比为1%~5%聚乙二醇辛基苯基醚(Triton X-100)的溶液;
脱细胞工作液Ⅲ为质量百分比为0.5%~5%脱氧胆酸钠的溶液;
脱细胞工作液Ⅳ为质量百分比为0.01%~3%过氧乙酸、1%~8%乙醇的溶液。
9.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S5中酶解时,添加比例为10mg脱细胞颗粒:1mg胃蛋白酶:1mL 0.01M盐酸溶液,使得脱细胞颗粒最终浓度为10mg/mL,消化15~30h。
10.根据权利要求1所述的一种具有生物活性的温敏角膜修复水凝胶的制备方法,其特征在于:步骤S6中,防腐体系为0.1%~0.5%无水三氯叔丁醇、0.1%~0.3%羟苯乙酯、0.015-0.2%羟苯甲酯、0.002%~0.01%苯扎氯氨中的任一种。
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